Background:

Learning Objective:

• Evaluate how Direct In Sample RNA Sequencing enables multiomic applications through integrated transcriptomic and phenotypic profiling

Direct in sample RNA sequencing enables measurement of gene expression directly within cells and tissues, linking genotype to phenotype at high resolution. Our approach preserves cellular and spatial context, allowing simultaneous imaging of cell morphology (e.g., Cell Painting) and RNA molecules targeted for sequencing. Here, we present two complementary approaches: a 3′ transcriptome method using poly-T probes to capture polyadenylated mRNA, and a targeted strategy employing probes custom designed against specific RNA features of interest. Both leverage a single-sided probe chemistry for cDNA extension, circularization, and rolling-circle amplification, followed by sequencing via ABC chemistry.

We applied these methods across diverse experimental contexts. First, in Optical Pooled Screening (OPS), we used direct RNA sequencing to link CRISPR-based perturbations with transcriptome profiling, multiplexed protein detection (50-plex), and high-content imaging (six cell-painting channels). This multimodal approach revealed perturbation-linked changes across modalities, highlighting the power of this technology in functional genomics.

Next, using the 3’ transcriptome approach, we profiled gene expression changes across variable cellular types and states, enabling direct, library and barcode-free quantification of differential gene expression at subcellular spatial resolution. Further, direct sequencing and alignment of targeted sequences (up to 100bp) enabled precise detection of alternative splicing and RNA aberrations within complex mixtures, offering molecular specificity beyond standard spatial transcriptomic assays.

These results demonstrate the versatility of Direct In Sample RNA Sequencing as a promising tool for integrated molecular profiling across a range of biological systems.